Human non-pancreatic secretory phospholipases A2 (sPLA2) are enzymes of 14 kD in size that effect ester hydrolysis of phospholipids at the sn-2 (or 2-acyl) position of 3-sn-phosphoglycerides to release a lysophospholipid and a fatty acid (ruzanski, W., et al, Immunology Today, 1991, 12, 143-146; Dennis, E. A., Trends Biochem., Sci., 1997, 22, 1-2; Murakami, M., et al, J. Biol. Chem., 274, 44, 31435-31444 Predominantly, the fatty acid released is arachidonic acid. When arachidonic acid is produced, it may be acted upon by other enzymes, including cyclooxygenases and lipoxygenases and transformed into inflammatory mediators such as platelet activating factor, prostalgandins, thromboxanes, lipoxins, lysophosphatides and leukotrienes (see Pnizanski supra). The presence of elevated levels of sPLA2 enzymes at sites of injury or inflammation has been demonstrated in a wide range of diseases and inflammatory conditions and the levels of enzyme activity have been correlated with the severity of the pathology (see Pruzanski supra). Compounds that inhibit the activity of sPLA2 might therefore prevent the formation of inflammatory mediators and could potentially alleviate symptoms associated with inflammatory conditions.
There are at least nine published mammalian sPLA2 enzymes including the human enzymes:—
Type IBpancreasType IIasynoviocytes, plateletsType IIdspleenType Vmast cell, macrophage, heart, lungType Xthymocytes, spleen, leukocytes
Recently, abnormally high concentrations of human non-pancreatic secretory PLA2-type IIa (hnpsPLA2) were detected in synovial fluid of humans with rheumatoid arthritis and osteoarthritis, and in the blood of humans suffering from bums, sepsis, psoriasis, Crohn's disease, adult respiratory distress syndrome (ARDS), acute pancreatitis, bacterial peritonitis, asthma, malaria, atherosclerosis, cancer, pregnancy complications and post-operative states. In all cases, the severity of the disease strongly correlated with elevated sPLA2 levels, suggesting a role for sPLA2 in immune defence. Prolonged excessive sPLA2 levels can, however, be deleterious, due to continuous eicosanoid formation and proteolytic activity. Intravenous administration of hnpsPLA2 to rabbits, for example, produces symptoms of arthritis and sepsis.
It has also been shown that types IIa and V produce arachidonic acid from several sources and that endogenous substances, membrane disturbances and disease processes promote such cleavage. There is a growing body of work which suggests that a key finction of hnpsPLA2 (type IIa) is an acute-phase protein, and a major bactericidal component of human tears, that helps eliminate infectious organisms and damaged host cells during the inflammatory response. Interestingly, it is also reported that this enzyme, which has quite a charged hydrophilic surface, normally has little activity in vitro on uncharged phosphatidylcholine vesicles and cell membranes. Mutation of residue 3 to the more hydrophobic residue Trp (Trp3) appears to increase binding by the PLA2 to, and hydrolysis of, such lipid surfaces by 2-3 orders of magnitude, consistent with the notion that the indole side chain of Trp3 helps penetrate the lipid interface of membrane substrates. The interfacial characteristics and membrane processing rates of the Trp3 mutant of hnpsPLA2 resemble those of mammalian pancreatic enzymes that also have a Trp at position 3. In addition to these surface effects on enzymatic function, very recently it has been proposed that some of the physiological functions that are regulated by sPLA2 are mediated by specific interactions between PLA2 surface residues and cellular receptors and, although there is evidence for this, the surface roles for PLA2 are not at all clear. Thus, while enzymatic activity of hnpsPLA2 is believed to stimulate neutrophils to produce superoxide, its surface properties are thought to be responsible for release of elastase and other degradative agents. To summarize, there is mounting evidence, therefore, that sPLA2 enzymes participate extensively in regulating not just phospholipid digestion, but also both transcellular and intracellular conununications involved in diverse physiological functions as well as in disease development.
Purported inhibitors of various phospholipases A2, particularly non-humanPLA2 enzymes have been described. However, it is now recognised that difficulties associated with the assay methods previously used have led to many of these compounds incorrectly being attributed with the desired activity. Many of these compounds are now recognised as not being potent inhibitors and generally do not inhibit human non-pancreatic secretary PLA2 enzymes at submicromolar concentrations (Balsinde, J., Ann, Rev. Pharmacol. Toxiol., 1999, 39, 175-89).
Thus, there exists a continued need for new inhibitors of sPLA2 which may be useful in the treatment of inflammatory diseases and conditions.